The long-term objective of this proposal is to study the mechanism by which steroid hormones cause the transformation of their receptors from an inactive form to a form that binds to DNA and is active in regulating gene transcription. The work is divided into two foci, with one focus being the study of the physical changes in the receptor complex that accompany glucocorticoid receptor (GR) transformation and the second focus being the study of receptor phosphorylation as it is related to receptor function. Glucocorticoids are used to treat a wide variety of diseases, including several forms of cancer. Understanding the mechanism by which these hormones cause their receptor mediated effects constitutes a fundamental problem in molecular endocrinology. During the past three years on this grant, we have shown that the untransformed GR is associated with the 90-kDa heat shock protein and that the hormone promotes dissociation of the GR from hsp90 during the transformation event. Hsp90 is a component of the 9S receptor complex which appears to be derived from the inactive state of the GR in intact cells. We have recently shown that, in the intact cell, hsp90 is associated with microtubules. This proposal presents a series of experiments designed to determine if the GR of L cells associates with tubulin-containing structures in cytosol, to determine the stoichiometry of components of the untransformed receptor complex, and to study the relationship between hsp90 dissociation and acquisition of DNA-binding activity in a system in which they inactive heteromeric GR complex has been immunopurified. We have recently shown that the phosphorylation state of the GR in L cells does not change with transformation and have mapped the endogenous phosphorylation sites with respect to the functional domains of the receptor. We will now determine if receptor that has been transformed and translocated to the nucleus in a hormone-dependent manner in the intact cell undergoes phosphorylation as part of the nuclear cycle of events. We know that the 15-kDa tryptic GR fragment containing the DNA-binding domain is phosphorylated and we will determine in the immunopurified Gr system whether this phosphorylation modifies the ability of the GR to bind DNA in a sequence-specific manner.